Protease assay for therapeutic drug monitoring

ABSTRACT

The present invention concerns a further development and use of biological assays to determine the amount or concentration of an active ingredient present in a sample. The enzyme assay of the present invention determines the amount or concentration of protease inhibitors, including retroviral protease inhibitors such as HIV inhibitors.

Qualitative and quantitative assays are of great importance in differentfields of the life sciences. The present invention concerns the use ofbiological assays to determine the amount or concentration of an activeingredient present in a sample. In particular, the enzyme assay of thepresent invention determines the amount or concentration of proteaseinhibitors, including retroviral protease inhibitors such as humanimmunodeficiency virus protease inhibitors, in a sample.

Therapeutic drug monitoring plays an important role in follow-up oftreatment efficacy. In order to achieve a therapeutic effect, drugsacting as enzyme inhibitors should be administered in dosages thatprovide sufficient inhibition of the enzyme involved. Diagnostic toolshave been developed to monitor the therapeutic effect of drugs in apatient at a certain concentration.

Current day monitoring assays for HIV (human immunodeficiency virus,HIV) inhibitors rely on techniques such as thin layer chromatography(TLC), high pressure liquid chromatography (HPLC), mass spectrometry(MS) and liquid chromatography-mass spectrometry (LC-MS) [Ther. DrugMonit. 2001, 23(4), 380; J. Chromatogr. B Biomed Sci. Appl (2001),758(2), 129; J. Chromatogr. B Biomed Sci. Appl (2000), 740(1), 43].Though these methods can monitor simultaneously several inhibitors in asingle patient sample, these assays do not provide a measure ofinhibition of the target enzyme. Leela et al. report that currentmethods for determining HIV inhibitors are not routinely available in aclinical laboratory and that a simplified, less tedious assay is neededand still needs to be developed. [Ann. Pharmacother. 2001, 35(6), 745].

The use of enzymes to monitor drug concentrations was described in e.g.U.S. Pat. No. 4,918,001, U.S. Pat. No. 4,546,076, U.S. Pat. No.5,576,177 and WO 99/54734. The latter patent publication discloses theuse of coated pins that are inserted into a solution. For instance, ifthe pins are coated with a substrate, then the protease and eventuallythe inhibitor are present in the solution. Inserting the pins starts thereaction, and removal of the pins from the reaction vessel stops theenzymatic reaction. U.S. Pat. No. 4,546,076 discloses the use of anenzyme assay to monitor the concentration of β-lactam antibioticspresent in a sample. U.S. Pat. No. 4,918,001 describes an assay tomeasure the concentration of endogenous protease inhibitors.

Methods have been described in which the residual protease activitypresent in a patient sample is measured [see e.g. EP148193; Ther. DrugMonit. 2001, 23(2), 93]. The residual enzyme activity is a measure ofthe drug effect and patient compliance. This type of assay providesinformation whether the pathway is sufficiently inhibited. Noinformation regarding the level of said drug in the sample is obtained.This type of assay can only be used for routine monitoring if the targetenzyme is readily accessible in a non-invasive manner and is present insaid sample in sufficient quantities to enable an enzymatic reaction.Therefore, this approach cannot be readily used for screening HIVprotease inhibitors present in a patient sample such as serum, since HIVprotease is not present in sufficient quantities in serum.

The use of fluorescent substrates to measure the HIV protease activitywas described by e.g. Matayoshi et al. [Science 1990, 247, 954], Tyagiet al. [Anal. Biochem. 1992, 200(1), 143], Toth et al. [Int. J. Pept.Protein Res. 1990, 36(6), 544] and Wang et al. [Tetrahedron 1990,31(45), 6493] and in several patent publications [see e.g. WO99/67417;EP428000, EP518557]. These disclosures describe how to determine theeffect of inhibitors on the HIV protease. These assays are useful forhigh throughput screening. Whereas in high throughput strategies apositive negative screening, i.e. inhibition or not, is performed,clinical assays need to provide a measure of therapy effectiveness inorder to enable the physician to draw conclusions related to thetherapy. In screening assays, the concentration of the compound underinvestigation is known, however, the compound concentration is unknownin clinical samples.

In view of the individualization of patient therapy, there is a need fora flexible approach to determine the amount or concentration of anactive ingredient in a biological sample. In addition, there is a needfor an assay that also can account for the effect of metabolites ofdrugs on the bio-active molecule. Therefore, the present invention wasmade, comprising adding the bio-active molecule to a biological sample,and measuring the activity of the bio-active molecule. In view of theimportance to determine the concentration of free and protein bounddrugs, the present invention introduces a separation procedure to meetthis need.

The instant invention relates to an improved bio-analytical method todetermine drug concentrations present in a patient sample. Morespecifically, the method concerns a fluorescent assay to quantify theinhibitory potency of a biological sample. The method may be used in anintegrated approach towards therapeutic drug modeling, linking resultsof bio-analytical techniques with resistance tests and pharmacologicaldata.

The invention relates to a method for determining the inhibitory potencyof an active ingredient in a biological sample, comprising: i) providinga biological sample; ii) providing a bio-active molecule; iii) providinga reagent for the bio-active molecule; iv) adding the biological sample,the bio-active molecule and the reagent for a bio-active molecule to acontainer; v) determining a signal; vi) relating the signal of v) to areference standard curve prepared with at least one reference.

According to the present invention a “biological sample” includes anysample derived from an organism i.e. a human or animal, optionallycomprising an active ingredient. A biological sample includes but is notlimited to blood, serum, plasma, saliva, cerebrospinal fluid, ejaculate,mammary ductal lavage and hair. A biological sample further includessamples obtained from culture flasks, wells, and other types ofcontainers. Said biological sample may comprise one or more activeingredients.

A “bio-active molecule” used in the present invention includes wild typebio-active molecules or recombinant versions thereof. The recombinantversions may include mutations indicative of resistance to one or moreactive ingredients, may include fluorescent or fluorescence quenchingmoiety(ies) or may be a fusion construct. The bio-active molecule can beused in conjunction with labeled substrates or ligands in order toprovide a homogeneous assay (e.g. fluorescence resonance transfer, FRET;bioluminescence resonance transfer, BRET). A bio-active molecule of thepresent invention means any biological molecule exhibiting for exampleenzyme activity e.g. HIV protease, HIV reverse transcriptase, HCVprotease (HCV, hepatitis C virus), HCV polymerase, matrix metalloproteinases, renin, thrombin; binding activity or other type ofinteraction. A bio-active molecule may be provided in a solution e.g. abuffered solution.

A “reagent” for a bio-active molecule includes substrates for enzymes;ligands for receptors. A reagent further includes, co-factors,antibodies, aptamers etc. In one embodiment the reagent is a substratefor HIV protease or a substrate for HCV protease. In one aspect of theinvention, the conversion of said substrate by the bio-active moleculemay be monitored using fluorescence. The reagent may be provided in asolution. Examples of solutions include buffers, organic solvents,buffered solutions completed with organic solvents.

An “active ingredient” means any compound, including a chemical, drug,antibody, ligand, antisense compound, aptamer, ribozyme, peptide,non-natural peptide, protein, PNA (peptide nucleic acid) and nucleicacids or a composition including at least one compound. This furthercomprises the compounds as administered and their metabolites.Metabolites may be generated under physiological conditions. As used inthe present invention the level of an active ingredient means the amountor concentration of said active ingredient in said sample. The activeingredient present in the biological sample may differ from the activeingredient in the reference. Under these circumstances, the inhibitorypotency of the biological sample equals an amount or concentration ofactive ingredient derived from a reference standard curve. The amountcan be expressed as for example, g, ml, mol. The concentration can beexpressed for example as ml/ml, g/l, M and the like.

A “matrix” according to the instant invention includes a solution, abuffer, a biological sample including but not limited to blood, plasma,serum, saliva, cerebrospinal fluid, ejaculate, mammary ductal lavage ortissue homogenates or a biological sample further completed with abuffer.

A “container” as used in the invention includes but is not limited to avessel, a well, a cuvette, a recipient etc. The compounds or liquids maybe mixed in said container. Suitably, the determination is performed insaid container. The container can be a separate entity e.g. a quartzcuvette or can be arranged in a multiple format such as e.g. a 16 wellplate, a 96 well plate, a 384 well plate.

A “signal” means any output generated by the biological assay includingfluorescence, fluorescence polarization, time resolved fluorescence,luminescence, time resolved luminescence, absorbance, radioactivity,resonance energy transfer mechanisms, magnetism. In one aspect, theoutput is generated during the assay. Examples of signals include,absorbance, relative fluorescence units and the like. The signal may begenerated by reacting the bio-active molecule with the reagent for thebio-active molecule. A signal may be determined at the end of apredefined incubation period, e.g. after 10 minutes. A signal may bemonitored continuously. This includes that a regular time intervals asignal is recorded. This regular time interval includes for instance atpredefined time intervals e.g. every 10 seconds, every 5 seconds and thelike. A signal may be converted to a ratio of signals. Examples of suchratios include the ratio of a signal generated by a biological sampleover the signal generated in a control reaction. A signal may beconverted to kinetic variables including initial velocity, maximalvelocity, mean velocity.

A “reference” means an active ingredient used to prepare the referencestandard curve. The reference is a single compound or a mixture of atleast two compounds. Said reference may be present in an identicalmatrix as the biological sample. Said reference does not necessarilyneed to be the same active ingredient as present in the biologicalsample. In one approach the compound used as a reference is the same asthe active ingredient present in the biological sample.

A “biological assay” means an assay relying on determining thebiological effects generated by a bio-active molecule including thedetermination of an enzyme reaction, a receptor ligand interaction, DNApolymerase activity, reverse transcriptase activity, integrase activity,antigen-antibody interactions and the like.

“Relating” means that the signal determined, is compared to a referencestandard curve, from which curve, the signal can be converted to ameasure of inhibition or to a level of reference compound.

“Reference standard curve” includes but is not limited to a curvewherein the signal is plotted against the measure of inhibition. Saidmeasure of inhibition may be expressed as a percentage. The referencestandard curve further includes curves in which the signal is plottedagainst a concentration of reference or an amount of reference. Areference standard curve may also be constructed by plotting a ratio,e.g. a ratio of the signal determined with the biological sample overthe signal determined with the control, against a level of an activeingredient. Suitably, said reference standard curve has been preparedaccording to the procedure of the present invention in particular asrecited steps i) to v). Upon preparing a reference standard curve, areference instead of a biological sample is used in step i). Thebio-active molecule and reagent for a bio-active molecule, however, areidentical to those used for the analysis of the biological sample.“Corresponding value” includes signal, activity, percentage ofinhibition, level of active ingredient present in the reference based onthe determination of the reference in the reference standard curve. Saidlevel may be expressed as a concentration (e.g. Molar, g/l, g/g etc.) oras an amount (mol, g, ml, mg, . . . ).

The “inhibitory potency” may be expressed as a percentage of inhibition,as a level of active ingredient i.e. amount or concentration of activeingredient. It is an object of the instant invention that the activeingredient present in the biological sample may differ from the activeingredient used for establishing a reference standard curve. If theactive ingredient used for preparing the reference standard curvediffers from the active ingredient in the biological sample, theinhibitory potency of the biological sample may be related to anequivalent amount of active ingredient used for preparing the referencestandard curve. In such case, the inhibitory potency may be expressed asa drug level, i.e. the signal generated by a biological sample will berelated to a corresponding drug level. In one embodiment, the activeingredient used for preparing the reference standard curve is the sameas the active ingredient present in the biological sample. In thislatter case, the inhibitory potency is equivalent to the level of theactive ingredient in the biological sample.

Upon determining the inhibitory potency of an active ingredient in abiological sample, the steps i) to iii) may be put in any order. In stepiv), the respective components may be added in different orders. Forinstance, if a protease activity is determined according to the methodsof the instant invention, three components are needed, a biologicalsample, a protease, and a substrate for the protease. These threecomponents may be brought together in any order, provided that for thepurpose of the present invention, the reaction is started with eitherthe bio-active molecule or the reagent for the bio-active molecule. Forinstance, the biological sample may be first added to the reagent of thebio-active molecule. In this procedure the reaction is started by addingthe bio-active molecule to the mixture, and subsequent determination ofthe signal. It is clear that also the biological sample may be firstadded to the bio-active molecule. In this approach, the reaction isstarted by addition of the reagent for the bio-active molecule. It willbe clear to the person skilled in the art that in the above procedureinstead of adding the biological sample to any of the bio-activemolecule or reagent of the bio-active molecule, the reverse may also beperformed. For instance, the bio-active molecule may be added to thebiological sample, after which the reaction mixture is completed with areagent of the bio-active molecule.

A blank signal may be determined. This means a reaction withoutbio-active molecule, e.g. the enzyme, or without reagent for thebio-active molecule. For these purposes, the bio-active molecule or thereagent for the bio-active molecule may be substituted by buffer. Saidblank signal may be subtracted from the signal determined when preparingthe reference standard curve, or when determining the inhibitory potencyof an active ingredient in a biological sample. Said blank signalaccounts for the signal generated by the reagent without interactionwith the bio-active molecule or reagent for the bio-active molecule.

A control signal may be determined. Said control signal is obtained whenno active ingredient is present in the biological sample or in thematrix to prepare the reference standard curve. Said control signal maybe used as a 100% value. Alternatively said control signal may be usedto obtain ratios of the signals generated with biological samples orwith the references to prepare the reference standard curve.

According to one approach, the biological sample is treated with asolvent, such as an organic solvent including methanol, ethanol, toobtain an extract comprising active ingredients. The extract, i.e. thesolution comprising active ingredients, is subsequently transferred to acontainer. Subsequently, in the case of HIV, the inhibitory potency maybe determined by measuring the activity of the HIV protease aftercompleting the container with the protease and a substrate for theprotease. Both, the extract and the biological sample may need dilutionprior to the assay. In one aspect, the biological sample or extract maybe diluted to obtain a signal which ranges from about 30% to about 70%of the signal of the control, interestingly from about 40% to about 60%of the signal of the control.

The protease can be a wild-type form or contain one or more mutations.Those mutations may influence the protease activity. In one embodiment amutant HIV protease may be used. For instance the HIV protease maycomprise one or more mutations selected from the list consisting ofmutations at amino acid position 3, 10, 20, 24, 32, 33, 35, 36, 37, 46,47, 50, 53, 54, 57, 58, 63, 70, 71, 72, 73, 77, 82, 84, 88, 89 or 90vis-à-vis wild type HIV protease (Genbank accession K 03455). Theprotease can be present in a buffered solution. A buffered solution isused to maintain the pH of the solution constant during the reaction andmay be further completed with ions to adjust the ionic strength of thebuffer and may be further completed with e.g. anti-oxidants, metalchelators, detergents, co-factors or solvents. Examples of buffersinclude citrate buffer, phosphate buffer, acetate buffer. The proteasemay be present in a concentration ranging from about 1 picomolar toabout 10 micromolar, suitably ranging from about 1 nanomolar to about 1micromolar, during the biological assay.

The substrate can also be provided in a solution, a buffered solution,an organic solvent or mixtures thereof. In one approach, the same buffermay be used to prepare the protease solution and the substrate solution.The enzyme activity may be measured using for instance a fluorogenic,quenched fluorogenic or chromogenic substrate. Other methods to measureinteractions between a bio-active molecule, its reagent and activeingredient are known in the art and may rely e.g. on FRET, BRET. Inthese latter approaches, one or both of the interacting compounds may belabeled by a fluorescent molecule. The interaction results in aresonance transfer of the excited wave length. Substrates which are ofinterest in view of the present invention include those compoundsincluding a moiety selected from the group consisting of7-amino-4-methylcoumarin; 7-amino-4-carbamoylcoumarin, paranitroanilide,beta-naphtylamide, aminobenzoyl, tetramethylrhodamine, EDANS, DANSYL,fluorescein or DABCYL. The substrate may contain a peptide backbonederived from HIV proteins including gag, protease, reversetranscriptase, integrase, p17, p24 or combinations thereof.Interestingly, said peptide backbone contains 2 to 30 amino acids,suitably 3 to 15 amino acids, more suitably, 3 to 10 amino acids.Examples of such backbones include the sequences S-Q-N-T-P-I-V-N,S-Q-N-Y-P-I-V-W-L or S-Q-N-Y-P-I-V-Q-K, wherein the amino acids arerepresented by their one letter code (Practical Biochemistry, 5^(th)edition, Ed Wilson&Walker, Cambridge University Press, Cambridge, 2000,p 313). An interesting backbone comprises the amino acid sequence P-I-V.Another interesting backbone comprises the tetrad Y-P-I-V. Aninteresting substrate is A-R-V-Y-F(NO₂)-E-A-Nle (Nle, norleucine).Self-quenching substrates may also be used (WO 99/50579). Interestingsubstrates to measure HIV protease include those comprising combinationsof DABCYL and EDANS in a peptide backbone; or the combination ofaminobenzoyl and nitrophenyl moieties in a peptide backbone. In case HCVprotease is determined, substrates may be designed based on the sequenceof HCV proteins. Interesting substrates include:(7-methoxycoumarin-4-yl)acetyl(Mca)-D-D-I-V-P-C-S-M-S-(2,4-dinitrophenyl,Dnp) K and Mca-D-D-1-V-P-C-S-M-K(Dnp)-R-R (J. Virol. Meth. 1999, 80,77-84), Ac-D-T-E-D-V-V-P(Nva)-O-4-phenylazophenyl ester (Ac,acetyl)(Nva, norvaline)(Anal. Biochem. 1999, 270, 268-275), internallyquenched depsipeptide fluorogenic substrates based on resonance energytransfer between the donor/acceptor couple5-[(2′-aminoethyl)amino]naphthalene sulfonicacid/4-[[4′-(dimethylamino)phenyl]azo]benzoic acid. Interestingsubstrates for hepatitis C virus protease are based on resonance energytransfer of depsipeptide substrates (Anal. Biochem. 1996, 240, 60-67).During the biological assay, the substrate may be present m aconcentration ranging from about 1 nanomolar to about 500 millimolar,interestingly ranging from about 1 micromolar to about 100 millimolar,more interestingly, from about 10 micromolar to about 1 millimolar.

The method for determining can be performed at room temperatureobviating the need for temperature controlled incubating infrastructure.Room temperature includes temperatures in the range from about 17° C. toabout 30° C. In one approach, this ranges from about 20° C. to about 26°C. In one approach, the reaction is run at about 25° C. However, theassays can also be performed at other temperatures e.g. in the range ofabout 30° C. to about 45° C., in one embodiment between about 35 toabout 40° C. or at about 37° C.

The period for incubating may be up to 1 day. In one approach theincubation period is short and ranges from about 15 seconds to about 2hours, interestingly said period ranges from about 30 seconds to about60 minutes. During said incubation period the signal may be monitored.Said signal may be monitored continuously, at predefined time intervalsor at a single time interval.

The volume during the incubation may be up to 10 ml. An interestingvolume ranges from about 10 μl to 5 ml, suitably from about 20 μl toabout 2 ml, more suitably from about 25 μl to about 500 μl. In amicrotiter plate an interesting volume ranges from about 25 μl to about300 μl.

The signal of the biological sample may be compared with data on areference standard curve. Said reference standard curve may be obtainedby measuring the signals of a variety of reference samples eachcomprising a defined drug concentration. Said reference may be presentin a matrix such as a solution, a buffer, an organic solvent and thelike. In one aspect the reference is present in the same matrix as thebiological sample. Said reference standard curve may be prepared usingdifferent references, each containing the same active ingredient but ata different concentration. Said reference standard curve may be preparedusing at least one reference, suitably, at least two references, eachhaving a different concentration of active ingredient; more suitably atleast three references, each having a different concentration of activeingredient. In one aspect the reference standard curve is prepared using4 to 8 different references of the same active ingredient, each having adifferent concentration. The active ingredient used for obtaining areference standard curve can be the same as the active ingredientpresent in the biological sample. By using a reference standard curve,the method of the current invention makes it possible to relate thegenerated signal to a specific concentration or amount of activeingredient present in the sample. This reference standard curve can beprepared using any compound or mixture of compounds. Consequently, theinhibitory potency of an active ingredient in a biological sample is ameasure of the total inhibitory capacity of a biological sampleirrespective the active ingredients present and irrespective their modeof interaction. For enzyme inhibitors said mode of interaction includesirreversible inhibition or reversible inhibition including competitive,non-competitive, anti-competitive, tight-binding or mixed inhibition.Based on such a reference standard curve, an estimate of the level ofactive ingredient(s) present in a biological sample, irrespective of theknowledge of the nature of the active ingredients, can be determined.This approach can be helpful for e.g. toxicology by indicating that agiven biochemical pathway is blocked with an equivalent of a knownactive ingredient.

Examples of compounds which may be analyzed according to the methods ofthe instant invention include dextran sulfate, suramine, polyanions,soluble CD4, PRO-542, BMS-806, T20, T1249, 5-helix, D-peptide ADS-J1,AMD 3100, AMD-3465, AMD7049, AMD3451, TAK 779, SHC-C(SCH351125), SHC-D,PRO-140, RPR103611, foscarnet and prodrugs, AZT, 3TC, DDC, DDI, D4T,Abacavir, FTC, DAPD, dOTC, DPC 817, PMEA, PMPA (tenofovir), nevirapine,delavirdine, efavirenz, 8 and 9-Cl TIBO (tivirapine), loviride, TMC-125,dapivirine, MKC-442, UC 781, UC 782, Capravirine, DPC 961, DPC963,DPC082, DPC083, calanolide A, SJ-1366, TSAO, 4″-deaminated TSAO, MV150,MV026048, SP1093V, PD126338, RO-5-3335, K12, K37, L 708906, L 731988,S-1360, anprenavir and prodrug GW433908 (VX-175, fosamprenavir),ritonavir, nelfinavir, saquinavir, indinavir, lopinavir, palinavir, BMS186316, atazanavir, DPC 681, DPC 684, tipranavir, AG1776, mozenavir,GS3333, KNI-413, KNI-272, L754394, L756425, LG-71350, PD161374,PD173606, PD177298, PD178390, PD178392, PNU 140135, TMC-114, maslinicacid, U-140690, castanospernine, deoxynojirimycine, CGP64222. Thesecompounds may also be included in a kit. Suitably, if HIV protease isused, the active ingredient may be selected from the list comprising:amprenavir, fosamprenavir, ritonavir, nelfinavir, saquinavir, indinavir,lopinavir, palinavir, BMS 186316, atazanavir, DPC 681, DPC 684,tipranavir, AG1776, mozenavir, GS3333, KNI-413, KNI-272, L754394,L756425, LG-71350, PD161374, PD173606, PD177298, PD178390, PD178392, PNU140135, TMC-114.

Using this assay, monitoring of the reaction kinetics may be performedin real time. Using this approach the steady state variables, e.g.initial velocity may be calculated (Practical Biochemistry, 5^(th)edition, Ed Wilson & Walker, Cambridge University Press, Cambridge,2000, Chapter. 7, p. 357-402). In one embodiment, the reaction isstopped before determining the signal. This is an end-pointdetermination. A reaction may be stopped by adding an agent including anorganic solvent, detergent, acid, base or by temperature changes. Theoutput e.g. fluorescence can be recalculated to parameters describingthe reaction e.g. initial velocity, or to measures of inhibition e.g.percentage inhibition or percentage residual activity.

The assay may also be used to determine concurrently 2 or more activeingredients, present in a biological sample, targeted against 2 or morebio-active molecules. For this purpose, 2 or more different substratesmay be used, each of which can be measured independently. Based on 2 ormore reference standard curves, the two or more active ingredients maybe quantified. For example, the inhibitory potency of biological samplecomprising both an HCV protease inhibitor and an HIV protease inhibitormay determined in a single well, using two different substrates. Onesubstrate is specific for HIV protease and one substrate is specific forHCV protease.

A further advantage of the present method is that it allows determiningthe amount of active ingredients bound to plasma proteins. Manycompounds are heavily protein bound, limiting their therapeuticefficacy. HIV protease inhibitors may be protein bound. An example of anHIV protease inhibitor binding protein is α-acid glycoprotein (AAG). Thesample may be treated to separate the free from the protein bound activeingredients. This separation can be performed by filtration. Filtrationmay be performed using gels e.g. gel filtration, filters, filter papers,using special microtiter plates. Other techniques are available in theart such as dialysis, heating, precipitation, centrifugation, antibodiesand other means known to the person skilled in the art. Alsocombinations of said techniques may be used to separate the proteinbound from the unbound active ingredient. The differentiation of thefree over the protein bound fraction may be important for compoundswhich are subject to protein binding and in conditions wherein theprotein concentration or the concentration of a particular proteinvaries over time in a bodily fluid. The knowledge of the distribution ofthe compounds between free and bound may be important in evaluating atherapy, especially if only the active ingredient is accessible to itstarget.

Since the bio-active molecule is added to the reaction mixture, thebio-active molecule does not necessarily need to be present in thesample. The method of the present invention further provides an assayfor those instances where the bio-active molecule is not readilyaccessible or is present in too low a concentration in a biologicalfluid like e.g. serum which may contain HIV inhibitors but only a verylow amount if any of the protease. Alternatively, the method may be usedfollowing removal from the biological sample of the bio-active moleculeconcerned or following inactivation of the bio-active molecule. Theselatter approaches may be valuable in those instances where theconcentration of said bio-active molecule varies between individuals.Removal or inactivation may be achieved by different methods includingbut not limited to precipitation by organic solvents, salts, detergents,change of pH or thermal denaturation.

In one approach, the assay of the instant invention is homogenous, has ashort turnover time and facilitates the parallel processing of multiplesamples. The assay of the instant invention can be run in a cuvettebased format or in multi-well format e.g. a microtiter plate.

The assay design is such that it determines the presence i.e. the levelof active ingredients inhibiting the enzyme in biological samples and isespecially suited for monitoring active ingredients in individuals undertreatment as well as in individuals subject to clinical trials. Thedetermination of the concentration can provide evidence whether thedosage of the compound is high enough to provide a therapeutic effect.

The instant invention also relates to a kit comprising a bio-activemolecule, a reagent for said bio-active molecule, and optionally anactive ingredient. The bio-active molecule, the reagent, and the activeingredient may be present in separate containers. The bio-activemolecule, the reagent, and the active ingredient may be lyophilized. Thekit may be further completed with a container comprising a buffer forthe enzymatic reaction. Said buffer may be provided as a lyophilizedpowder. If the kit is composed of lyophilized components, said kit maybe further completed with one or more solutions for the dissolution ofsaid lypohylized powders. The kit may contain one or more containerscomprising one or more active active ingredients. The containercomprising the active ingredient, needed to prepare a reference standardcurve, may optionally contain the protease or the substrate for theprotease.

Another level of complexity in diseases like cancer and HIV, is thefrequent occurrence of resistance towards treatment. These latterconditions require a frequent monitoring of drug levels in conjunctionwith resistance testing. Therefore, methods have been developed whichmonitor the phenotypic alterations in the population of HIV virionscirculating in the patient (e.g. WO 97/27480). Other phenotyping assaysinclude those described by Witvrouw (WO 01/57245), Virologic(WO97/27319) and Bioalliance (WO 02/38792). The Antivirogram®(WO97/27480) estimates the drug susceptibility of the viral population,as compared to ‘wild-type’ strains. In this test service, the drugconcentration that inhibits virus growth for 50% (IC₅₀) is determined invitro. The ratio IC₅₀ of the virus in a patient's blood sample over theIC₅₀ of ‘wild-type’ virus is the fold change in drug susceptibility ofthe virus in that patient. An alternative for the phenotypic assays inwhich the patient borne material is grown under in vitro conditions, isgenotyping. Genotyping assays estimate the occurrence of resistancebased on sequence variations. One improvement over the genotypic assaysis called VirtualPhenotyping (WO 01/79540) in which the genotype of apatient virus is compared to a collection of genotypes present in adatabase and for which the corresponding phenotypes are stored in adatabase. These assays provide an accurate determination of the drugeffect and the occurrence of resistance of the virus in a patient, yetno information on circulating drug levels are provided. Therefore, it isan aspect of the instant invention to link drug level determinations,according to the methods of the present invention, to resistancetesting. Linking the determination of drug levels to resistance testingcan provide additional evidence of the therapy efficacy.

The minimum plasma concentration or trough concentration can be criticalduring treatment of diseases or conditions wherein the drug target issubject to modulation in order to overcome the drug effect. Examples ofthis latter phenomenon are found in infectious diseases like HIV,bacterial infections and cancer. For example, the presence of drugsgenerates mutational pressure on the HIV protease to escape from drugtherapy and insufficient inhibitor concentration facilitates suchescape. The determination of the drug level in a patient and the use ofthis value to determine the trough level can be important in obtainingdosages high enough to yield a therapeutic effective concentration.

The concentration of an active ingredient, determined using theprocedure of the instant invention, can be used in pharmacologicalmodels to provide estimates of pharmacokinetic parameters describing thedrug profile in an individual. Using population pharmacokinetic models,the trough drug level can be determined from a single patient sample.Suppose a large group of HIV-infected patients receive the sameantiretroviral drug in the same dose three times daily. The averageplasma concentration-time profile of the drug in the patient populationmay look as shown in FIG. 1 (bold line). However, due to theinter-individual variability of pharmacokinetic processes (absorption,distribution, elimination), individual curves may substantially differfrom the typical profile, as exemplified by the dotted line. If allindividual curves are plotted, they may cover a range marked by thevertical bars. If on the same graph, individual minimum effectiveconcentrations (MECs) (dashed horizontal line gives an example, FIG. 1)are provided, they will also cover some range. The drug concentration ina fraction of the patients may drop below their MEC and this reduces thetherapeutic outcome, and possibly leads to drug resistance. The troughlevel of an individual patient can subsequently be used to calculate thedrug level required to attain therapeutic effective doses during thedosing interval.

The methods and results of the instant invention, e.g. the concentrationof a drug, may be used to determine pharmacological parameters of thedrug in an individual, including trough levels (C_(t)), maximalconcentration (C_(max)), the area under the curve (AUC), eliminationvelocity etc. Therefore, the determined level may be inputted in apopulation pharmacokinetic model and pharmacokinetic variables such asC_(t), C_(max), AUC may be calculated (WO 02/23186). Thesepharmacological variables may be used to estimate for example thepotential toxicity of a certain dose, exposure time to a drug (e.g. incase of radiochemicals) or minimum concentration found.

In order to obtain effective treatment, the exposure of an individual toa drug e.g. trough concentration, AUC, must exceed a certain level. Thislevel is determined by the nature of the virus population. The ratio ofthe exposure to the drug (trough level, AUC, other) over thedrug-resistance (fold-resistance, IC₅₀, IC₉₀, other) is predictive forsuccessfulness of the therapy. This ratio can be expressed as the IQ(inhibitory quotient) (IQ=C_(t)/IC₅₀.). This IQ value can be furthernormalized in order to obtain a value adjusted for protein binding(normalized IQ, NIQ). One approach of obtaining this adjusted value isto determine the mean C_(t) for a population, and the IC₅₀ for an activeingredient for a reference strain i.c. a reference laboratory HIVstrain. The quotient of these latter two values yields(C_(t)/IC₅₀)_(reference). The normalized IQ is provided by the quotient:[(C_(t)/IC₅₀)]_(patient)/[(C_(t)/IC₅₀)]_(reference).

In one embodiment, the instant invention provides a method fordetermining the inhibitory quotient (IQ), wherein the trough level isbased on the level of the active ingredient present in a biologicalsample determined according to the methods described herein. The IQ canbe further normalized.

The invention further provides a method of designing a therapy, themethod comprising: determining the level of an active ingredient presentin a biological sample according to the methods described herein,determining the trough level for said active ingredient, recalculatingthe dosage for said active ingredient using a population kinetic modelto achieve active ingredient levels exceeding the minimal effective doseduring the dose interval.

In a further development the concentration found in the patient samplemay be transmitted electronically to a server where the pharmacokineticvalues are determined through population based methods. These valuesi.e. pharmacokinetic data and drug levels may be linked to resistancedata to provide an integrated measure of therapy efficacy. The providedinformation may be further used to design a therapy in order to achievesufficient clinical effect. These data may, following processing, betransferred back to e.g. the treating physician.

The assay may be used for analysing drug levels in animals andpharmacokinetic studies in animals.

In one aspect, the present invention concerns a method of determiningthe inhibitory potency of a biological sample, the method comprising:(a) obtaining a biological sample, providing a container comprising atarget bio-active molecule, adding the biological sample to saidcontainer to obtain a mixture, adding a reagent of the bio-activemolecule to the mixture and determining a signal, (b) relating thesignal to a reference standard curve prepared with at least onereference sample, wherein the reference sample has the same matrix asthe biological sample, the matrix optionally completed with an activeingredient at a defined concentration, and wherein the signal of thereference sample has been determined according to (a).

The assay procedure can be used to monitor interactions between enzyme,substrate and inhibitor but equally well between receptor and ligand;recepor and antagonist; receptor and agonist; antigen and antibody. Forthe analysis of active ingredients interacting with receptor molecules,ligands instead of substrates are preferred for the analysis method ofthe present invention.

In one embodiment, the assay of the instant invention adds to the art aflexible, fast, easy to standardize, economical and homogenous assay tomonitor drug levels in patient samples using an enzyme. Multiple assayscan be run in parallel and inter-laboratory variation can be limited byproviding kits containing the required elements to perform the reaction.

FIGURES

FIG. 1: Plasma concentration of active ingredient (mg/l) as a functionof time. MEC is the minimum effective concentration. The time isexpressed in hours. The plasma drug concentration should vary betweenthe MEC and the toxic level.

FIG. 2: Monitoring of substrate cleavage by HIV protease in real time ina fluorescence microtiter plate reader. Each square (A1 tot H10)represents the progress curves of the fluorescence (Y-axis) as afunction of time (X-axis). The assay was run as described in theexperimental part. The slope of each curve represents the initialvelocity of the enzymatic reaction. Row A contains control samplesWithout inhibitor. Columns 1 and 2 contain extracts of dog serum spikedwith different concentration of compound 1 used to generate acalibration curve. Columns 3 and 4; 5 and 6; 7 and 8; 9 and 10, containdog serum spiked with 100 nM, 10 nM, 10,000 nM, and 1,000 nM of compound1,2 and 3 respectively. Row B to H contain dilutions of the spiked serumas indicated at the right. The dilutions vary from 1:120 (row B) to 1:1(row H). See example 1 for details.

FIG. 3: Initial rate of substrate cleavage calculated using linearregression by plate reader software. The substrate cleavage wasmonitored in real time in a fluorescence microtiter plate reader.Extracts of dog serum spiked with different concentrations of compound 1were used. The relative fluorescence units (RFU, Y-axis) were monitoredas a function of time (seconds, X-axis).

FIG. 4: Calculation of a reference standard curve, the data furtherillustrate the accuracy of the captioned method. Sa: sample;concentration in nM; Values: in (RFU/min)×1000; StDev (standarddeviation): in (RFU/min)×1000; CV %: coefficient of variation;MeanValue: mean Value in (RFU/min)×1000; Ratio: ratio of the MeanValueof Sa0X over the MeanValue of Sa01. X is 2 to 8 (see figure).

FIG. 5. Typical reference standard curve relating the concentration ofinhibitor in nM (X-axis) to the ratio of test sample compared to acontrol sample (expressed as a ratio of RFU values, Y-axis)

FIG. 6: Calculation of analyte concentration. The calculation of theanalyte is performed automatically by the plate-reader software.Concentration was calculated for analyte presented in columns 9 and 10on FIG. 2. The data show that samples may need to be diluted prior toanalysis. In the present case a dilution between 6 to 60 is optimal.(StDev=Standard deviation; Adj. Result=result adjusted for dilution).

FIG. 7: Accuracy and precision of the method. Five independentexperiments were performed on five different dates to assess theaccuracy and reproducibility of the method.

Exp.: experiment. The error of each determination is shown inparenthesis and represents the difference between measured inhibitorconcentration and spiked inhibitor concentration expressed in %.

EXAMPLES

The present example and accompanying drawings illustrate the presentinvention. Their illustrative purpose is not construed as limiting thescope of the invention.

Example 1 Determination of HIV PR Inhibitor (Human ImmunodeficiencyInhibitor, HIV; PR, Protease), Concentration in Serum Samples

The following compounds were used in the present analysis(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl-N-[(1S,2R)-3-[[(4-aminophenyl)sulfonyl](2-methylpropyl)amino]-2-hydroxy-1-(phenylmethyl)propyl]-carbamate,(compound 1, CAS 206361-99-1);(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl-N-[(1S,2R)-3-[(1,3-benzodioxol-5-ylsulfonyl)(2-methylpropyl)amino]-2-hydroxy-1-(phenylmethyl)propyl]-carbamate(compound 2, CAS 333798-27-9) and(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl-N-[(1S,2R)-2-hydroxy-3-[[(4-methoxyphenyl)sulfonyl](2-methylpropyl)amino]-1-(phenylmethyl)propyl]-carbamate(compound 3, CAS 206362-00-7). The method of the present invention mayalso be used with other sulfonamides as described in U.S. Pat. No.5,843,946. Four aliquots of normal dog serum were spiked with HIV PRinhibitor compound 1, to final concentration of 10, 100, 1,000 and10,000 nM (referred later as analyte samples) respectively. Another 8aliquots of dog serum were used to generate a reference standard curve.These 8 aliquots were spiked with different concentration of compound 1from 0 to 100 nM. Thus compound 1 was used as reference to prepare areference standard curve. Serum proteins in all samples wereprecipitated using 66% (v/v) methanol (MeOH). To one part of serumsample two parts of MeOH were added, mixed and centrifuged at 16,100 gfor 30 min. The supernatant was recovered and can be stored for at least2 weeks at −20° C. prior to assay. 60 μl is used for the cuvette assayor 24 μl is used for the plate reader assay. Six serial dilutions 1:2 to1:120 of the extracts, obtained from analyte samples were prepared priorto assay. Methanol extract of normal dog serum without inhibitor wasused for the dilution.

For the plate reader assay, the following conditions were used: Thebuffer was prepared of 50 mM Sodium acetate (NaOAc), pH 4.5, 2 mMdithiothreitol (DTT), 200 mM Sodium Chloride (NaCl), 0.01% Tween 20. Thesubstrate was present at a final concentration of 20 μM in the abovebuffer. The substrate used is based on a natural substrate for HIVprotease. It contains a portion (P₄-P₄′) of the p17-p24 gag protein.R-E(EDANS)-S-Q-N-Y-P-I-V-Q-K(DABCYL)-R-OH displays only lowfluorescence, yet upon cleavage by HIV protease at Y-P bond fluorescenceaugments (Science, 1989, 247, 954-958). The enzyme, wild type HIV-1protease, was present in the above buffer (50 mM NaOAc, pH4.5, 2 mM DTT,200 mM NaCl), 0.01% Tween 20) at a final concentration of 4-5 nM. Itshould be appreciated that other forms of HIV protease can be used aswell, including mutant forms of the protease.

The assay is performed at room temperature (RT). 88 μl of buffer (whichcontains the enzyme) were dispensed and 24 μl of serum extract or serumextract dilution was added using multi-channel pipettor. The reactionwas started by adding 88 μl buffer containing substrate and the increaseof fluorescence was monitored over time (FIG. 2). The reference standardcurve was prepared according to this method using 24 μl of referenceinstead. The control reaction consisted of extracted serum void of anyprotease inhibitor. Initial rate of substrate cleavage, that wascalculated by the plate reader software for each well (FIG. 3) wasinverse proportional to inhibitor concentration. The initial rate ofcontrol sample provided a 100% signal and was used to determine theratio with respect to the samples from the reference standard curve aswell as from analyte samples, containing an inhibitor (ratio signalsample/signal control).

The initial rate of fluorescence increase was monitored for eachcompound 1 dilution (FIG. 2, columns 1 and 2) to yield a referencestandard curve (FIG. 4, 5). The reference standard curve links the ratio(signal sample/signal control) to the drug concentration for eachcompound 1 dilution. The measure of inhibition, expressed as a ratio,provides an estimate of the corresponding concentration of compound 1.For example, the concentration of inhibitor in analyte sample presentedin columns 9 and 10 on FIG. 2, can be calculated using plate readersoftware as shown on FIG. 6. For the final calculation twoconcentrations that gave the ratio closest to 0.5 were averaged. Theresulted calculated concentration was 1,109 nM, comparable to actualconcentration in this sample—1,000 nM. Note that the reference standardcurve can be prepared using any protease inhibitor or mixture thereof.

Though, the above assay does not discriminate between different HIVprotease inhibitors, it provides a reliable and accurate determinationof HIV protease inhibition and concurrently the level of the inhibitoryactivity in the biological sample. This assay format determines thetotal amount of protease inhibitor present in a biological sample. Inaddition, the assay can provide evidence on the percentage of proteinbound inhibitor as exemplified in Example 3.

Determination of inhibitor concentration as described in Example 1 wasperformed on five different days to evaluate precision and accuracy. Theresults are presented on FIG. 7.

The principle of the present invention can be used to any type ofcompound interfering with a bio-active molecule. It will apparent thatthe person skilled in the art will know different approaches of workingthe instant invention.

Example 2 Effect of MeOH

The methanol extraction is commonly used for determination of HIV PRinhibitor concentrations in human serum by LC-MS. Serum proteins areprecipitated with 75% MeOH. Since high concentration of methanol caninterfere with enzyme-based assays, the MeOH concentration used for theextraction of inhibitors was reduced to 66%. Following test wasperformed to compare the extraction with 66% MeOH and 75% MeOH. Mixturesof compound 1 and 2 were prepared at 9 μM and 0.9 μM concentrations andextracted with 66 and 75% MeOH as described in example 1. The resultsare summarized in Table 1. “Ext” means extraction in Table 1. TABLE 1Control 66% Ext 75% Ext Inhibitor (in μM) % error (in μM) % Error (inμM) % Error   9 μM 8.94 0.6 10.5 16.6 8.34 7.3 0.9 μM 0.85 5.5 0.99 100.77 14.4

Example 3 Determination of Protein Bound Versus Total Drug Concentration

According to multiple studies most of HIV PR inhibitors are highlyplasma protein bound (e.g. 90-99% for amprenavir, nelfinavir, saquinavirand ritonavir and about 60% for indinavir (IDV)). Since protein bindingaffects the drug potency, a simple method for the determination ofplasma protein bounded versus unbounded drug concentration wasdeveloped. Following protocol was used to measure the unbound drugconcentration:

550 μl of normal human serum was spiked with the protease inhibitorSC-52151 at final concentration 1 μM. 50 μl aliquot was used for thetotal inhibitor concentration determination as described in example 1.SC-52151 was used to generate a calibration curve.

500 μl spiked serum sample was loaded on microcon-10 microconcentratorand spun @5000×g, at room temperature for 5 min. The flow throughfraction (˜10% of load, 50 μl) was extracted with 66% methanol accordingto the protocol described in example 1. The filtrate contains theunbound fraction of protease inhibitors. Different dilutions of methanolextract were prepared and the micro titer plate reader assay wasperformed to determine unbound or free drug concentration as describedin example 1. As in the case of total inhibitor concentration (example1), the unbound drug concentration is presented as an inhibitoryactivity equivalent to reference drug concentration (in this case theinhibitory activity equivalent to a SC-52151 concentration in nM derivedfrom the calibration curve).

Using the methods of the instant invention the ratio of the free versustotal drug concentration can be determined and expressed as apercentage. TABLE 2 Percentage of HIV protease inhibitor in sample notbound to proteins Sample (spiked with 1 μM drug) IDV SC-52151 Free/totaldrug ratio (%) 36.5 10.5

The free indinavir concentration (36.5%) is in good agreement with datapresent in the literature (40%). In human serum, HIV protease inhibitorsare primarily bound to α-acid glycoprotein. The free drug concentrationdetermined in these experiments is inversely proportional to the bindingconstant of the drug to alpha-acid glycoprotein. K_(a) is the bindingconstant. IDV SC-52151 K_(a) for AAG binding (10⁶ M⁻¹) No binding 2.3

1. A method for determining the inhibitory potency of an activeingredient in a biological sample, comprising: i) providing a biologicalsample; ii) providing a bioactive molecule; iii) providing a reagent forthe bio-active molecule; iv) adding the biological sample, thebio-active molecule and the reagent for a bio-active molecule to acontainer; v) determining a signal; vi) relating the signal of v) to areference standard curve prepared with at least one reference.
 2. Amethod according to claim 1, wherein the bio-active molecule is aprotease and the reagent for the bio-active molecule is a substrate. 3.A method according to claim 2, wherein the biological sample comprisesan HIV inhibitor as active ingredient, the protease is HIV protease, andthe substrate is a substrate for HIV protease.
 4. A method according toclaim 3, wherein the substrate comprises an EDANS and/or DABCYL moiety.5. A method according to claim 4, wherein the substrate isR-E(EDANS)-S-Q-N-Y-P-I-V-Q-K(DABCYL)-R-OH (SEQ ID NO: 10).
 6. A methodaccording to claim 3, wherein the HIV protease comprises at least onemutation.
 7. A method according to claim 1, wherein the signal influorescence.
 8. A method according to claim 1, wherein the container isa well in a multi-well plate.
 9. A method according to claim 1comprising, wherein prior to step iv) the protein bound and proteinunbound fraction of the active ingredient in the biological sample areseparated.
 10. A method according to claim 1 comprising, wherein priorto step iv) the protein bound and protein unbound fraction of the activeingredient in the biological sample are separated.
 11. A methodaccording to claim 10, wherein the protein bound and protein unboundfraction of the active ingredient in the biological sample is separatedthrough a process selected from precipitating, filtrating, dialysis andcentrifuging.
 12. A method according to claim 1 wherein the biologicalsample comprises a reversible inhibitor.
 13. A method according to claim1, wherein the reference for determining the reference standard curvehas the same biological matrix as the biological sample.
 14. A methodaccording to claim 1, wherein the inhibitory potency is expressed asdrug concentration or as a drug amount.
 15. A method for determining atrough level of a drug in an individual using a populationpharmacokinetic model, characterized in that the drug concentration ordrug amount is determined according to claim
 14. 16. A method fordetermining the inhibitory quotient of a drug in a biological sample,using the trough level of the drug in an individual determined accordingto claim
 14. 17. A method for designing a therapy, comprising:determining the trough level of a drug in an individual according toclaim 15, recalculating the dosage for the drug using a populationkinetic model, achieving drug levels exceeding the minimal effectivedose during the dose interval.
 18. A kit, comprising a bio-activemolecule at least one active ingredient, a reagent for the bio-activemolecule.
 19. A method for determining the inhibitory potency of anactive ingredient in a biological sample, the method comprising: (a)obtaining a biological sample, providing a container comprising abio-active molecule, adding the biological sample to said container toobtain a mixture, adding R-E-(EDANS)-S-Q-N-Y-I-V-Q-K(DABCYL)-R-OH (SEQID NO: 10) of the HIV protease to the mixture and determining afluorescence signal, (b) relating the signal to a reference standardcurve prepared with at least one reference sample, wherein the referencesample has the same matrix as the biological sample, the matrixoptionally completed with an active ingredient at a definedconcentration, and wherein the signal of the reference sample has beendetermined according to (a).
 20. A method according to claim 2, whereinthe signal in fluorescence.
 21. A method according to claim 2, whereinthe container is a well in a multi-well plate.
 22. A method according toclaim 2 comprising, wherein prior to step iv) the protein bound andprotein unbound fraction of the active ingredient in the biologicalsample are separated.
 23. A method according to claim 2 comprising,wherein prior to step iv) the protein bound and protein unbound fractionof the active ingredient in the biological sample are separated.
 24. Amethod according to claim 2 wherein the biological sample comprises areversible inhibitor.
 25. A method according to claim 2, wherein thereference for determining the reference standard curve has the samebiological matrix as the biological sample.
 26. A method according toclaim 2, wherein the inhibitory potency is expressed as drugconcentration or as a drug amount.